Plant genetic engineering projects require access to a variety of genetic elements that are used to regulate transgene expression. Two examples of such genetic elements are promoters and introns.
Initiation of transcription is regulated by a promoter. A given project typically requires use of several different promoters. One promoter will be used to drive the gene of interest, and a different one used, for example, to drive the selectable marker.
A eukaryotic gene is usually interrupted by noncoding sequences called introns. The initial product of transcription of a eukaryotic gene is pre mRNA, which includes sequences corresponding to the introns. The introns are removed during post-transcription processing to provide the mRNA that is translated to produce a protein. Studies characterizing the role of introns in the regulation of gene expression have shown that the first intron of the maize alcohol dehydrogenase gene (Adh-1) has the ability to increase expression under anaerobiosis. Callis J., M. Fromm, and V. Walbot. (1987), Gene Dev. 1:1183–1200. The intron also stimulates expression (to a lesser degree) in the absence of anaerobiosis. This enhancement is thought to be a result of a stabilization of the pre-mRNA in the nucleus. Mascarenhas et al. reported a 12-fold and 20-fold enhancement of CAT expression by use of the Adh-1 intron. Mascarenhas et al., “Intron-Mediated Enhancement of Heterologous Gene Expression in Maize,” Plant Molecular Biology, 15:913–920, 1990. Several other introns have been identified from maize and other monocots which increase gene expression. Vain, P. et al. (1996), Plant Cell Reports 15:489–494. See also WO98/5921.
The cDNA sequence for maize ADF is known (GenBank accession no. X97726), but the genomic ADF sequence has not been published. In particular, the sequence for the ADF promoter has not heretofore been published, nor have any ADF introns been identified.